In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics

Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes.This paper has the objectiv of to analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. It found that the mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. How main conclusions, we have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development

Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1(142–153) and Art v 1(25–36), respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals

miRNA–target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity